Yawei Gao

Distinct features of H3K4me3 and H3K27me3 chromatin domains in pre-implantation embryos.

Histone modifications have critical roles in regulating the expression of developmental genes during embryo development in mammals. However, genome-wide analyses of histone modifications in pre-implantation embryos have been impeded by the scarcity of the required materials. Here, by using a small-scale chromatin immunoprecipitation followed by sequencing (ChIP–seq) method, we map the genome-wide profiles of histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 27 trimethylation (H3K27me3), which are associated with gene activation and repression, respectively, in mouse pre-implantation embryos. We find that the re-establishment of H3K4me3, especially on promoter regions, occurs much more rapidly than that of H3K27me3 following fertilization, which is consistent with the major wave of zygotic genome activation at the two-cell stage. Furthermore, H3K4me3 and H3K27me3 possess distinct features of sequence preference and dynamics in pre-implantation embryos. Although H3K4me3 modifications occur consistently at transcription start sites, the breadth of the H3K4me3 domain is a highly dynamic feature. Notably, the broad H3K4me3 domain (wider than 5 kb) is associated with higher transcription activity and cell identity not only in pre-implantation development but also in the process of deriving embryonic stem cells from the inner cell mass and trophoblast stem cells from the trophectoderm. Compared to embryonic stem cells, we found that the bivalency (that is, co-occurrence of H3K4me3 and H3K27me3) in early embryos is relatively infrequent and unstable. Taken together, our results provide a genome-wide map of H3K4me3 and H3K27me3 modifications in pre-implantation embryos, facilitating further exploration of the mechanism for epigenetic regulation in early embryos.

Identification of key factors conquering developmental arrest of somatic cell cloned embryos by combining embryo biopsy and single-cell sequencing

Differentiated somatic cells can be reprogrammed into totipotent embryos through somatic cell nuclear transfer. However, most cloned embryos arrest at early stages and the underlying molecular mechanism remains largely unexplored. Here, we first developed a somatic cell nuclear transfer embryo biopsy system at two- or four-cell stage, which allows us to trace the developmental fate of the biopsied embryos precisely. Then, through single-cell transcriptome sequencing of somatic cell nuclear transfer embryos with different developmental fates, we identified that inactivation of Kdm4b, a histone H3 lysine 9 trimethylation demethylase, functions as a barrier for two-cell arrest of cloned embryos. Moreover, we discovered that inactivation of another histone demethylase Kdm5b accounts for the arrest of cloned embryos at the four-cell stage through single-cell analysis. Co-injection of Kdm4b and Kdm5b can restore transcriptional profiles of somatic cell nuclear transfer embryos and greatly improve the blastocyst development (over 95%) as well as the production of cloned mice. Our study therefore provides an effective approach to identify key factors responsible for the developmental arrest of somatic cell cloned embryos.

SIRT6 Controls Hematopoietic Stem Cell Homeostasis through Epigenetic Regulation of Wnt Signaling.

Proper regulation of Wnt signaling is critical for the maintenance of hematopoietic stem cell (HSC) homeostasis. The epigenetic regulation of Wnt signaling in HSCs remains largely unknown. Here, we report that the histone deacetylase SIRT6 regulates HSC homeostasis through the transcriptional repression of Wnt target genes. Sirt6 deletion promoted HSC proliferation through aberrant activation of Wnt signaling. SIRT6-deficient HSCs exhibited impaired self-renewal ability in serial competitive transplantation assay. Mechanistically, SIRT6 inhibits the transcription of Wnt target genes by interacting with transcription factor LEF1 and deacetylating histone 3 at lysine 56. Pharmacological inhibition of the Wnt pathway rescued the aberrant proliferation and functional defect in SIRT6-deficient HSCs. Taken together, these findings disclose a new link between SIRT6 and Wnt signaling in the regulation of adult stem cell homeostasis and self-renewal capacity.

The Combination of Tet1 with Oct4 Generates High‐Quality Mouse‐Induced Pluripotent Stem Cells

The DNA dioxygenase Tet1 has recently been proposed to play an important role in the reprogramming of somatic cells to pluripotency. Its oxidization product 5‐hydroxymethylcytosine, formerly considered an intermediate in the demethylation of 5‐methylcytosine, has recently been implicated as being important in epigenetic reprogramming. Here, we provide evidence that Tet1 (T) can replace multiple transcription factors during somatic cell reprogramming and can generate high‐quality mouse induced pluripotent stem cells (iPSCs) with Oct4 (O). The OT‐iPSCs can efficiently produce viable mice derived entirely from iPSCs through tetraploid complementation; all 47 adult OT‐iPSC mice grew healthily, without tumorigenesis, and had a normal life span. Furthermore, a new secondary reprogramming system was established using the OT all‐iPSC mice‐derived somatic cells. Our results provide the first evidence that the DNA dioxygenase Tet1 can replace multiple pluripotency transcription factors and can generate high‐quality iPSCs with Oct4. Stem Cells 2015;33:686–698

Hierarchical Oct4 Binding in Concert with Primed Epigenetic Rearrangements during Somatic Cell Reprogramming.

The core pluripotency factor Oct4 plays key roles in somatic cell reprogramming through transcriptional control. Here, we profile Oct4 occupancy, epigenetic changes, and gene expression in reprogramming. We find that Oct4 binds in a hierarchical manner to target sites with primed epigenetic modifications. Oct4 binding is temporally continuous and seldom switches between bound and unbound. Oct4 occupancy in most of promoters is maintained throughout the entire reprogramming process. In contrast, somatic cell-specific enhancers are silenced in the early and intermediate stages, whereas stem cell-specific enhancers are activated in the late stage in parallel with cell fate transition. Both epigenetic remodeling and Oct4 binding contribute to the hyperdynamic enhancer signature transitions. The hierarchical Oct4 bindings are associated with distinct functional themes at different stages. Collectively, our results provide a comprehensive molecular roadmap of Oct4 binding in concert with epigenetic rearrangements and rich resources for future reprogramming studies.

Maternal Sall4 Is Indispensable for Epigenetic Maturation of Mouse Oocytes

Sall4 (Splat-like 4) plays important roles in maintaining pluripotency of embryonic stem cells and in various developmental processes. Here, we find that Sall4 is highly expressed in oocytes and early embryos. To investigate the roles of SALL4 in oogenesis, we generated Sall4 maternal specific knock-out mice by using CRISPR/Cas9 system, and we find that the maternal deletion of Sall4 causes developmental arrest of oocytes at germinal vesicle stage with non-surrounded nucleus, and the subsequent meiosis resumption is prohibited. We further discover that the loss of maternal Sall4 causes failure in establishment of DNA methylation in oocytes. Furthermore, we find that Sall4 modulates H3K4me3 and H3K27me3 modifications by regulating the expression of key histone demethylases coding genes Kdm5b, Kdm6a, and Kdm6b in oocytes. Moreover, we demonstrate that the aberrant H3K4me3 and H3K27me3 cause mis-expression of genes that are critical for oocytes maturation and meiosis resumption. Taken together, our study explores a pivotal role of Sall4 in regulating epigenetic maturation of mouse oocytes.

Reprogramming of H3K9me3-dependent heterochromatin during mammalian embryo development

H3K9me3-dependent heterochromatin is a major barrier of cell fate changes that must be reprogrammed after fertilization. However, the molecular details of these events are lacking in early embryos. Here, we map the genome-wide distribution of H3K9me3 modifications in mouse early embryos. We find that H3K9me3 exhibits distinct dynamic features in promoters and long terminal repeats (LTRs). Both parental genomes undergo large-scale H3K9me3 reestablishment after fertilization, and the imbalance in parental H3K9me3 signals lasts until blastocyst. The rebuilding of H3K9me3 on LTRs is involved in silencing their active transcription triggered by DNA demethylation. We identify that Chaf1a is essential for the establishment of H3K9me3 on LTRs and subsequent transcriptional repression. Finally, we find that lineage-specific H3K9me3 is established in post-implantation embryos. In summary, our data demonstrate that H3K9me3-dependent heterochromatin undergoes dramatic reprogramming during early embryonic development and provide valuable resources for further exploration of the epigenetic mechanism in early embryos.Gao and colleagues characterize genome-wide H3K9me3 distributions in pre- and post-implantation mouse embryos, providing a resource to further our understanding of epigenomic dynamics during mammalian embryogenesis.

Direct induction of neural progenitor cells transiently passes through a partially reprogrammed state

The generation of functional neural progenitor cells (NPCs) holds great promise for both research and clinical applications in neurodegenerative diseases. Traditionally, NPCs are derived from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), or NPCs can be directly converted from somatic cells by sets of transcription factors or by a combination of chemical cocktails and/or hypoxia. However, the ethical issues of ESCs, the risk of tumorigenesis from iPSCs and transgenic integration from exogenous genes as well as complicated manipulation and time-consuming of chemical induced NPCs (ciNPCs) limit the applications of these strategies. Here, we describe a novel method for generating growth factor-induced neural progenitor cells (giNPCs) from mouse embryonic and adult fibroblasts by using inductive and/or permissive signaling culture conditions. These giNPCs closely resemble brain-derived NPCs in terms of transcription networks and neural lineage differentiation potentials. Moreover, this somatic cell to NPC induction is a gradual process that includes initiation, intermediate, maturation and stabilization stages. Importantly, gene expression and histone modification analyses further indicate a partially reprogrammed state during the generation process of induced NPCs, in which lineage specific genes and pluripotency associated genes are transiently activated. Our study therefore describes the potential safety problems that also exist in the transgene-free direct induction strategy and highlights the importance of excluding the possibility of residual partially reprogrammed and/or teratoma-like cells from the generated NPCs for future clinical trials.

Baf60b-mediated ATM-p53 activation blocks cell identity conversion by sensing chromatin opening

Lineage conversion by expression of lineage-specific transcription factors is a process of epigenetic remodeling that has low efficiency. The mechanism by which a cell resists lineage conversion is largely unknown. Using hepatic-specific transcription factors Foxa3, Hnf1α and Gata4 (3TF) to induce hepatic conversion in mouse fibroblasts, we showed that 3TF induced strong activation of the ATM-p53 pathway, which led to proliferation arrest and cell death, and it further prevented hepatic conversion. Notably, ATM activation, independent of DNA damage, responded to chromatin opening during hepatic conversion. By characterizing the early molecular events during hepatic conversion, we found that Baf60b, a member of the SWI/SNF chromatin remodeling complex, links chromatin opening to ATM activation by facilitating ATM recruitment to the open chromatin regions of a panel of hepatic gene loci. These findings shed light on cellular responses to lineage conversion by revealing a function of the ATM-p53 pathway in sensing chromatin opening.

Protein Expression Landscape of Mouse Embryos during Pre-implantation Development

Pre-implantation embryo development is an intricate and precisely regulated process orchestrated by maternally inherited proteins and newly synthesized proteins following zygotic genome activation. Although genomic and transcriptomic studies have enriched our understanding of the genetic programs underlying this process, the protein expression landscape remains unexplored. Using quantitative mass spectrometry, we identified nearly 5,000 proteins from 8,000 mouse embryos of each stage (zygote, 2-cell, 4-cell, 8-cell, morula, and blastocyst). We found that protein expression in zygotes, morulas, and blastocysts is distinct from 2- to 8-cell embryos. Analysis of protein phosphorylation identified critical kinases and signal transduction pathways. We highlight key factors and their important roles in embryo development. Combined analysis of transcriptomic and proteomic data reveals coordinated control of RNA degradation, transcription, and translation and identifies previously undefined exon-junction-derived peptides. Our study provides an invaluable resource for further mechanistic studies and suggests core factors regulating pre-implantation embryo development.